Method and composition for treating periodontal disease

ABSTRACT

The present invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of misoprostol and an effective amount of an antibiotic. A suitable antibiotic is selected from the group consisting of doxycycline, gentamicin, tobramicin, ciprofloxacin, clindamycin, clarithromycin, azithromycin and metronidazole. Preferred antibiotics are doxycycline and ciprofloxacin. More preferably, the antibiotic is doxycycline. In its second aspect, the present invention is directed to a method for treating periodontal disease in a mammalian patient comprising administering to a mammalian patient in need of such treatment a therapeutically effective amount of misoprostol and an effective amount of an antibiotic. Typically, the mammalian patient is a human.

This application is a continuation of U.S. Ser. No. 10/635,144, filedAug. 6, 2003, now allowed.

FIELD OF THE INVENTION

The present invention is directed to pharmaceutical compositions and amethod for using such compositions to regenerate bone and tissue indental pockets

BACKGROUND OF THE INVENTION

Periodontitis is a chronic inflammatory response caused by bacterialplaque that has spread below the gum line. Starting in the early stagesas gingivitis, the later stages of periodontitis involves inflammationof the gums, connective tissues, and bones surrounding the teeth i.e.alveolar bones. Prolonged inflammation causes degenerative loss oftissues supporting the teeth and alveolar bone loss, eventually leadingto loss of teeth. Tooth loss, caused by the loss of alveolar bone, isone of the major problems in clinical dentistry. In fact, periodontitisis the primary cause of tooth loss. It is approximated that 140 millionadults, in the United States alone, exhibit various stages ofperiodontal disease.

The periodontitis-associated inflammation occurring in the surroundingtissues that support the teeth is characterized by formation of infected“pockets” or spaces between the teeth and gums. These infected pocketscontain debris, predominantly composed of microorganisms and theirproducts (enzymes, endotoxins and other metabolic products), dentalplaque, gingival fluid, food remnants, salivary mucin, desquamatedepithelial cells, and leukocytes. Periodontal pockets are chronicinflammatory lesions, and as such are constantly undergoing repair. Thecondition of the soft tissue walls of the periodontal pocket resultsfrom a balance between destructive and constructive tissue changes. Thedestructive changes consist of the fluid and cellular inflammatoryexudates and the associated degenerative changes stimulated by localbacterial infiltrate. The constructive changes consist of the formationof connective tissue cells, collagen fibers, and blood vessels in aneffort to repair tissue damage caused by the inflammatory process.Healing does not go to completion because of the persistence of localirritants i.e., bacteria and the enzymes that they produce. Theseirritants stimulate fluid and cellular exudates, which in turn causesdegeneration of the new tissue elements formed in the repair process. Ifpurulent exudates are present in the infected pockets, it can containliving, degenerated and necrotic leukocytes (predominantlypolymorphonuclear), living cells and dead bacterial cells, serum and asmall amount of fibrin.

The most basic treatment for periodontitis is scaling and root planingprocedures. These procedures involve manually removing calculus, plaqueand other deposits, smoothing the root surface to rid it of necrotictooth substances, and curetting the inner surface of the gingival wallof the periodontal pockets to separate away any diseased soft tissue.The procedures aim to eliminate the infected pockets by reattachingconnective tissue and epithelium to the tooth surface. By eliminatingthe environment for the microorganisms to grow, scaling and root planingprocedures can successfully obliterate the infected pockets. Theseprocedures may result in the replacement of diseased soft tissue withnew soft tissue from growth and differentiation of new cells andintercellular substances. However, scaling and root planing proceduresare ineffective in stimulating the re-growth or replacement of destroyedbone and cementum caused by severe periodontitis. Hence, there is a needfor reliable or predictable methods to regenerate, augment, or restorealveolar bone loss and cementum loss inflicted by periodontitis.

Another initial treatment for periodontitis is administration ofantibiotics, which may be optionally performed in conjunction withscaling and root planing procedures. Tetracycline is the most commonlyadministered antibiotic for periodontitis; however, numerous other typesof antibiotics can also be employed because human periodontal pocketsharbor highly diverse populations of bacteria. In fact, a recent studyby Paster et al. (Paster, B. J., Journal of Bacteriology, Vol. 183 (12),p. 3770-3790, 2001) recorded 91 different bacterial species orphylotypes from cultures collected from periodontal diseased sites inhuman patients. In those patients who suffered from refractoryperiodontitis, collected cultures revealed the presence of 213 differentbacterial species or phylotypes present. Novel bacterial species orphylotypes, in addition to known putative periodontal pathogens such asPorphyromonas gingivalis, Bacteroides forsythus, and Treponemadenticola, were observed in these cultures. In addition to tetracycline,other antibiotics, such as minocycline, as described in U.S. Pat. No.4,701,320 (the entire disclosure of which is incorporated herein byreference), and amoxicillin and metronidazole, as described in U.S. Pat.No. 4,997,830 (the entire disclosure of which is incorporated herein byreference), have been employed for the treatment of periodontitis.Despite the effectiveness of antibiotics in reducing inflammation andbacterial infection, antibiotic administration, like scaling and rootplaning, is also deficient in stimulating re-growth or replacement ofthe destroyed bone and cementum caused by severe periodontitis.

A more dramatic therapy for patients who are not responsive to scalingand root planing procedures and/or antibiotic administration isperiodontal surgery, such as gingivectomy or periodontal flap surgery.In gingivectomy, the dentist reshapes the unhealthy gum tissue in orderto reduce the size of the infected pocket. Reduction of the pocket sizeallows the patient to hygienically maintain the pocket by routinebrushing and flossing, thereby eliminating a favorable environment forbacterial growth. Periodontal flap surgery is performed also whenscaling and root planing procedures are unsuccessful, especially whenthere is loss of bone or tissue detachment. In this procedure, incisionsare made in the gums and the surrounding alveolar bone is re-contouredto assist in healing of the infected area. Since only one quadrant ofthe mouth can be operated on at a time, multiple visits to the dentistare required and the entire surgical procedure can extend over a periodof a month or longer. In addition to the invasive nature and theinherent pain involved with surgery, both procedures of gingivectomy andperiodontal flap surgery are insufficient in stimulating re-growth orreplacement of the destroyed bone and cementum caused by severeperiodontitis.

In an effort to address the problem of alveolar bone loss caused byperiodontitis, therapeutic compositions containing substances thatspecifically stimulate osteogenesis in mammalian skeleton have beendeveloped. One such substance is prostaglandin E1 (PGE₁). In vitrostudies, as described for example in Flanagan et al. (Flanagan, A. M. etal., Endocrinology, Vol. 130 (1), p. 443-448, 1992), have shown thatculturing mammalian calvarial cells in the presence of PGE₁ increasesthe formation of mineralized nodules. Formation of these nodules isadvantageous because they give rise to osteoprogenitor cells, which areresponsible for mediating the process of bone anabolism (Miller et al.,Clinics in Plastic Surgery, Vol. 21 (3), p. 393-400, July 1994). Therole of PGE₁ in bone formation has been confirmed by in vivo studiesperformed on adult dog models. A study by Marks et al. (Marks, S. C. etal., J. Oral Pathol., Vol 17, p. 500-505, 1988) shows that localapplication of PGE₁ can stimulate new bone formation adjacent to sitesof delivery in the canine mandible long bone and that local delivery ofPGE₁ increases the thickness and appositional rate of alveolar bone inthe canine mandible. A further study, also by Marks et al. (Marks, S. C.et al., J. Periodont. Res., Vol. 29, p. 103-108, 1994) shows that localdelivery of PGE₁ to the canine mandible increases alveolar bone heightand regenerates cementum and periodontal ligaments around the premolarsand molars of adult dogs. These in vivo studies demonstrate that PGE₁can promote osteogenesis and increase bone mass.

Recently, topical compositions comprising a synthetic and more stablePGE₁ analog, called misoprostol, have been developed as conjunctivetherapy with scaling and root planing procedures. As described, forexample in U.S. Pat. Nos. 5,324,746 and 5,510,384 (the disclosures ofwhich are incorporated herein by reference), these compositions areeffective in treating periodontal disease and can also be used to treatdamaged tissue caused by, for example, chemotherapy or radiotherapy.Other applications for compositions comprising misoprostol includeregeneration of collagen-containing human tissue, such as skin, bone,connective tissue, and cartilage, as described for example in U.S. Pat.No. 5,994,399 (the disclosure of which is incorporated herein byreference).

An object of the present invention is to provide a pharmaceuticalcomposition and methods that are more effective than misoprostol aloneor current methods in regenerating bone and cementum in periodontalpockets of patients afflicted with periodontal disease.

BRIEF SUMMARY OF THE INVENTION

The Applicants unexpectedly discovered a method for obtainingexceptional results in treating mammalian patients afflicted withperiodontal disease, particularly in obtaining very significantregeneration of bone and cementum in periodontal pockets as determinedby the very significant reduction in the depth of the periodontalpockets with or without surgical intervention (i.e., scaling, rootplaning and curettage). More specifically, the Applicants discoveredthat treating a mammalian patient afflicted with periodontal diseasewith a combination of an antibiotic that is effective against one ormore plaque causing bacterium that are found in a periodontal pocket(hereinafter an “antibiotic”) and with misoprostol produced anunexpectedly superior reduction in the depth of the periodontal pocketsin the afflicted patients. At the 6-week observation, the patientsreceiving the dual regimen, comprising systemically administering astandard dosing regimen of systemic antibiotic over the prescribeddosing period (typically 10 days) followed by topically applyingmisoprostol to the dental pocket(s), performed as well as the patientstreated with the single dosing regimen of antibiotic only. However, atthe 52-week observation, the patients receiving the dual regimen showedthe greatest reduction in pocket depth of all groups. The dual drugtreatment regimen was the only treatment that resulted in a trendtowards apparent continued improvement in pocket depth between the Week6 and the Week 52 observations. These patients had approximately 40-50%mean reductions in pocket depth at Week 6, and approximately 50-60%percent mean reduction at Week 52.

The most unexpectedly superior reduction in pocket depth was obtainedafter only three weeks of treatment when the patients afflicted withperiodontal disease were pretreated systemically with a standard dosingregimen of an antibiotic for 10 days followed by topically applying forthree weeks to, at, on, onto, in, into or near the periodontal pockets apharmaceutical composition comprising an effective amount of misoprostoland an effective amount of an antibiotic.

Thus, the present invention has multiple aspects. In one aspect, thepresent invention is directed to a pharmaceutical composition comprisingin combination a therapeutically effective amount of misoprostol and aneffective amount of an antibiotic. A suitable antibiotic is selectedfrom the group consisting of tetracycline, doxycycline, gentamicin,tobramicin, ciprofloxacin, clindamycin, clarithromycin, azithromycin andmetronidazole. Preferred antibiotics are doxycycline and ciprofloxacin.More preferably, the antibiotic is doxycycline. The pharmaceuticalcomposition typically utilizes one or more pharmaceutically acceptablecarriers for topical application in the mouth (also referred to hereinas “oral” application), and preferably, is applied in paste form, morepreferably utilizing a dental adhesive.

In its second aspect, the present invention is directed to a method fortreating periodontal disease in a mammalian patient comprisingadministering to a mammalian patient in need of such treatment atherapeutically effective amount of misoprostol and an effective amountof an antibiotic. Typically, the mammalian patient is a human. However,the term “mammalian patient” as used herein is intended to cover othermammals than humans. Such other mammals include mammals treated byveterinarians. Preferred other mammals include domesticated animals,such as dogs, cats, horses, cattle, sheep, goats, pigs and primates.

In one embodiment of the inventive method, the antibiotic isadministered to the mammalian patient separately from the misoprostol.In this embodiment, the antibiotic is administered systemically to thepatient whereas the misoprostol is topically applied to, at, on, onto,in, into or near the periodontal pocket. This topical administration isalso known in the art as “local” administration.

In another embodiment of the inventive method, the effective amount ofmisoprostol is topically applied in a pharmaceutical composition thatalso contains a topically effective amount of antibiotic. Preferably,the pharmaceutical composition that is used for the topicaladministration contains one or more pharmaceutically acceptable carriersthat are sufficiently adhesive so as to retain the composition as awhole at, on, onto, in, into or near the periodontal pocket. Suitablecarriers are known in the art and more fully discussed herein. It isalso within the scope of this latter embodiment that the patient besystemically pre-treated (i.e., treated prior to administration of themisoprostol) with a typical dosing regimen of antibiotic. As reflectedin the Physician's Desk Reference (PDR), a typical systemic dosingregimen for most antibiotics is oral administration over a period of 10days.

The present method for treating periodontal disease optionally includesthe step of administrating sub-gingival scaling, root planing, orcurettage in the afflicted patient. This step is optionally administeredprior to, during or immediately after systemically pretreating themammalian patient with an effective amount of antibiotic. Preferably,this step is administered prior to systemically treating the mammalianpatient with an effective amount of antibiotic.

In a particularly preferred embodiment, the present invention isdirected to a method for treating periodontal disease in a mammalianpatient comprising (a) administering to, at, on, onto, in, into, or neara periodontal pocket of the patient in need of treatment apharmaceutical composition comprising a therapeutically effective amountof misoprostol or a pharmaceutically acceptable derivative or saltthereof in a pharmaceutically acceptable carrier; (b) administering to,at, on, onto, in, into, or near a periodontal pocket of said patient inneed of treatment a pharmaceutical composition comprising an antibioticthat is effective against bacteria suspected of causing bacterial plaquein a pharmaceutically acceptable carrier; and (c) optionally, prior tostep (a) or step (b), performing sub-gingival scaling, root planing, orcurettage in said patient. Preferably, steps (a) and (b) are performedsimultaneously, more preferably, simultaneously in a singlepharmaceutical composition containing both the misoprostol andantibiotic.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

FIG. 1A is a comparative graph of the mean change in pocket depth (mm)per observation versus the baseline pocket depth (mm) at 6 weekspost-treatment for the following four treatment modalities: placebo(open circle); doxycycline (open triangle); doxycycline+misoprostol(shaded triangle); and misoprostol (shaded circle) for N=58.

FIG. 1B is a comparative graph of the mean change in pocket depth (mm)per observation versus the baseline pocket depth (mm) at 52 weekspost-treatment for the following four treatment modalities: placebo(open circle); doxycycline (open triangle); doxycycline+misoprostol(shaded triangle); and misoprostol (shaded circle) for N=40.

FIG. 1C is a comparative graph of the mean change in pocket depth (mm)per patient versus the baseline pocket depth (mm) at 6 weekspost-treatment for the following four treatment modalities: placebo(open circle); doxycycline (open triangle); doxycycline+misoprostol(shaded triangle); and misoprostol (shaded circle) for N=58.

FIG. 1D is a comparative graph of the mean change in pocket depth (mm)per observation versus the baseline pocket depth (mm) at 52 weekspost-treatment for the following four treatment modalities: placebo(open circle); doxycycline (open triangle); doxycycline+misoprostol(shaded triangle); and misoprostol (shaded circle) for N=40.

DETAILED DESCRIPTION OF THE INVENTION

The present invention has multiple aspects. In its first aspect, theapplicants have discovered a superior treatment for periodontal diseaseis achieved by a pharmaceutical composition comprising a therapeuticallyeffective amount of misoprostol and a therapeutically effective amountof an antibiotic in a pharmaceutically acceptable carrier.

The first active ingredient, misoprostol, is an analog of PGE₁ in whichthe carbon 15 (C-15) hydroxyl group has been removed and replaced by ahydrogen atom, and the two hydrogen atoms at the carbon-16 (C-16)position have been removed and replaced by a methyl group and a hydroxylgroup. By adding a methyl group to the C-16 position, the C-16 hydroxygroup becomes less susceptible to the action of 15-dehydrogenase enzymesthat inactivate natural prostaglandins. In addition, the C-16 positionof the hydroxyl group, rather than the 15-position as in natural PGE₁,reduces side effects, such as diarrhea, compared with the natural PGE₁.

Misoprostol, methyl-(11α,13E)-11,16-dihydroxy-16-methyl-9-oxoprost-13-en-1-oate, is racemic,existing as four stereoisomers in the 16R(±)- or 16S(±)-stereoisomericforms. Misoprostol is commercially available from the G. D. SearleDivision of Monsanto under the trade name CYTOTEC® as a double racemateof two diastereomers containing four stereoisomers. Structurally, thesestereoisomers are characterized by the structures shown below:

Misoprostol binds to both PGE₁ and PGE₂ receptors, although only one ofits four stereoisomers is thought to be responsible for mediatingreceptor activity. It is unknown as to which of the four stereoisomersis the active agent. Thus, it is reasonable to expect that some effect,such as those derived from misoprostol competing with PGE₁ at itsbinding site, for instance, will show significant stereospecificity.When administered, misoprostol is rapidly de-esterified to its activeform, misoprostolic acid. Misoprostolic acid is approximately 85%albumin bound and has a half-life of about 30 minutes, mostly beingexcreted in urine as inactive metabolites.

In addition to being used in topical compositions for the treatment ofperiodontitis, as described, for example, in U.S. Pat. No. 5,324,746(the entire disclosure of which is incorporated herein by reference),misoprostol has been used in the past on a prescription basis as agastric mucosa protectant and antiulcer agent. Misoprostol inducesmarked edema of the mucosa and submucosa (increasing the thickness ofboth layers), dilating interglandular regions of the lamina propria,reducing depth and width of gastric foveolae, vasodilating vascularchannels, reducing the height of surface epithelial cells, swellingbasolateral intercellular spaces, and increasing the amount of adherentmucosa. The mucosal edema and increased mucus layer may be importantcomponent in misoprostol's cytoprotective mechanism (Davies, N. M. etal., Pharmacotherapy, Vol. 21(1), p. 60-73, 2001). The primary use formisoprostol is the prevention of nonsteroidal anti-inflammatorydrug-induced (NSAID) gastric ulcers, and short term treatment ofduodenal ulcers. Additional diseases for which misoprostol is beingdeveloped as a viable therapeutic agent include, for instance,NSAID-induced small bowel damage, dysfunctional pancreatic enzymefunction and imbalanced duodenal pH caused by cystic fibrosis, andhepatic and pancreatic tissue injury and related GI damage (Shield, M.J. et al., Pharmac. Ther., Vol. 65, p. 125-147, 1995).

In its second aspect, the present invention is directed to a method fortreating periodontal disease comprising administering to a patient inneed of treatment a pharmaceutical composition comprising atherapeutically effective amount of misoprostol or a pharmaceuticallyacceptable derivative or salt thereof, and an effective amount of anantibiotic. As used herein, the term “therapeutically effective amountof misoprostol” is typically from about 1 microgram (μg) to about 40micrograms, preferably from about 5 micrograms to about 30 micrograms,more preferably from about 10 micrograms to about 20 micrograms, andmost preferably from about 10 micrograms to about 12 micrograms.According to the invention, misoprostol or pharmaceutically acceptablederivatives or salts thereof can be in the form of any pharmaceuticallyactive racemate, enantiomer, or diastereomer. In addition, anypharmaceutically acceptable misoprostol derivative, such asmisoprostolic acid, can be used in the method or compositions of theinvention.

Antibiotics that may be used in the compositions and method of theinvention include, but are not limited to, antibiotics selected from thegroup consisting of tetracycline derivatives, preferably doxycycline; anaminoglycoside, such as gentamicin or tobramicin; a fluoroquinolinederivative, such as ciprofloxacin; a lincomycin derivative, such asclindamycin; a macrolide derivative, such as clarithromycin; an azalidederivative, such as azithromycin; and an imidazole derivative, such asmetronidazole. By the term “a therapeutically effective amount of anantibiotic” is meant, for a given antibiotic, an amount of an antibioticthat effectuates stasis or a reduction in the overall bacterialpopulation in a periodontal pocket. In one embodiment of the presentinvention, the antibiotic was administered systemically so as to achievea therapeutically effective amount of antibiotic in the blood andultimately at the periodontal pocket. In a formulation that providesunexpectedly superior results and that facilitates patient compliance, atherapeutically effective amount of antibiotic was administeredtopically to, at, in, onto, into or near the periodontal pocket with atherapeutically effective amount of misoprostol. Preferably, theantibiotic and the misoprostol for topical application are containedwithin the same pharmaceutical composition.

In embodiments comprising systemically administered doxycycline, atherapeutically effective amount is know to the art and typically rangesfrom about 20-100 mg twice daily (BID) for one day (a loading dose) andfrom about 50-200 mg once each day (QD) for nine days, more typicallyfrom about 50-100 mg twice daily (BID) for one day and from about 50-100mg once daily (QD) for nine days, and most typically about 100 mg twicedaily (BID) for one day and about 100 mg once daily (QD) for nine days.For the topically administered doxycycline, a therapeutically effectiveamount comprises from 0.1 to 3 cc (ml) of a pharmaceutical compositionhaving from 0.1-5% of doxycycline, typically, about 0.1 to 3 cc of apharmaceutical composition having about 1% doxycycline. Similarformulations are made for tetracycline and minocycline.

In embodiments comprising systemically administered gentamicin ortobramicin, for example, a therapeutically effective amount of theantibiotic is known in the art. For the topically administeredgentamicin, a therapeutically effective amount comprises from 0.1 to 2cc (ml) of a pharmaceutical composition having from 0.1-5% ofgentamicin, typically, about 0.1 to 2 cc of a pharmaceutical compositionhaving from 0.1-1% of gentamicin, more typically, about 0.1 to 2 cc of apharmaceutical composition having about 0.1% gentamicin.

In embodiments comprising systemically administered ciprofloxacin, forexample, a therapeutically effective amount is known in the art andtypically ranges from about 250-700 mg twice daily (BID) for five to tendays, more typically for ten days. For the topically administeredciprofloxacin, a therapeutically effective amount comprises from 0.1 to5 cc (ml) of a pharmaceutical composition having from 0.1-5% ofciprofloxacin, typically, about 0.1 to 5 cc of a pharmaceuticalcomposition having from 0.1-1% of ciprofloxacin, more typically, from0.1 to 5 ml of a pharmaceutical composition having about 1%ciprofloxacin.

In embodiments comprising systemically administered clindamycin, forexample, a therapeutically effective amount is known in the art andtypically ranges from about 37.5-300 mg every 6-8 hours (3-4 timesdaily) for 5-10 days, more typically from about 150-300 mg every 6 hours(QID) for 5-10 days. For the topically administered clindamycin, atherapeutically effective amount comprises from 0.1 to 3 cc (ml) of apharmaceutical composition having from 0.1-5% of clindamycin, typically,about 0.1 to 3 cc of a pharmaceutical composition having about 1%clindamycin.

In embodiments comprising clarithromycin, for example, for example, atherapeutically effective amount is known in the art and typicallyranges from about 125-1000 mg once to twice daily (BID) for ten days,more typically from about 250-500 mg twice daily (BID) for 10-14 days or1000 mg once daily for 10-14 days. For the topically administeredclarithromycin, a therapeutically effective amount comprises from 0.5 to3 cc (ml) of a pharmaceutical composition having from 0.1-5% ofclarithromycin, typically, about 0.5 to 3 cc of a pharmaceuticalcomposition having about 2% clarithromycin.

In embodiments comprising azithromycin, for example, for example, atherapeutically effective amount is known in the art and typicallyranges from about 50-500 mg once daily for one day (a loading dose) andabout 250 mg once each day (QD) for four more days, more typically fromabout 500 mg once daily for one day and from about 250 mg once daily forfour more day. For the topically administered azithromycin, atherapeutically effective amount comprises from 0.1 to 3 cc (ml) of apharmaceutical composition having from 0.1-5% of azithromycin,typically, about 0.1 to 3 cc of a pharmaceutical composition having from0.5-2% of azithromycin, more typically, about 0.1-3 cc of apharmaceutical composition having about 1% azithromycin.

In embodiments comprising metronidazole, for example, for example, atherapeutically effective amount is known in the art and typicallyranges from about 250-2000 mg twice (BID) to four (QID) times daily forfive to ten days. For the topically administered metronidazole, atherapeutically effective amount comprises from 0.1 to 3 cc (ml) of apharmaceutical composition having from 0.1-5% of metronidazole,typically, about 0.1 to 3 cc of a pharmaceutical composition having from0.75-2% of metronidazole, more typically, about 0.1 to 3 cc of apharmaceutical composition having about 0.75% metronidazole.

According to the invention, the present pharmaceutical compositions maycomprise more than one antibiotic, such as a combination of antibiotics.Compositions comprising combinations of antibiotics for the treatment ofperiodontitis have been previously described. For example, U.S. Pat. No.4,997,830 (the disclosure of which is incorporated herein by reference)describes an oral composition comprising metronidazole and amoxicillinfor the treatment of periodontal disease.

Pharmaceutical compositions presented herein may be prepared in variousformulations to produce a variety of final products that are useful fortreating periodontal disease. For instance, for topical administrationto periodontal pockets, formulations such as toothpaste, mouthwash,chewing gum, and other dentifrices are embodiments of the pharmaceuticalcomposition of the invention. In addition, topical formulation products,such as gels, creams, lotions, ointments, and timed-release liposomesare also embodiments of the present invention. Topical and/or oralcompositions of this invention can be formulated with an appropriatechoice of one or more pharmaceutically acceptable carriers.Pharmaceutically acceptable carriers suitable for use with thisinvention include, for example, vegetable or mineral oils, whitepetrolatum or white soft paraffin, branched chain fats or oils, animalfats, and alcohols, such as absolute ethanol. Acceptable carriers mayalso be in soluble form. Emulsifiers, stabilizers and anti-oxidants mayalso be included as well as agents imparting color or fragrance ifdesired.

The pharmaceutical compositions of the present invention for topical(local) application may further comprise colloidal dispersion systems,such as carbowax; additives or preservatives, such as polyethyleneglycol having an average molecular weight ranging, for example, fromabout 300 MW to about 1450 MW; denture adhesives, such as SUPERWERNET'S® Denture Adhesive Powder (90% hypromellose, USP(2-hydroxypropyl methylcellulose) 10% polyox WSR-301 (polyethylene oxidewater soluble resin, MW approximately 4,000,000)); and/or a solvent,such as absolute ethanol or aqueous sodium chloride.

Oral pastes containing the present pharmaceutical compositions may beformulated by any method known. In an embodiment, 2.3 mg misoprostol(i.e., pulverized 11.5×200 microgram tablets of CYTOTEC® was combinedwith 15 grams of SUPER WERNET'S® Denture Adhesive Powder, 0.86 grams ofpolyethylene glycol (1450 MW), and 16.34 grams of polyethyleneglycol(300 MW). Use of the denture powder has been shown to allow a slowerrelease of the active agents from the application site on the oralmucosa. The oral paste (71 μg misoprostol/ml) delivers a dose of 10micrograms of misoprostol per application dose of 140 mg (0.12 ml) andcan be dispensed in two 10 ml syringes. The oral paste can be appliedover the area where the gingival tissue meets the tooth lingual, palataland buccal surfaces.

Oral washes containing the present pharmaceutical compositions may beformulated by any method known. In an embodiment, 0.06 ml of a solutionof misoprostol in absolute ethanol (400 micrograms of misoprostol perml) is diluted to 1.0 ml with 0.9% sodium chloride. The oral wash may beused for flushing infected pockets.

Creams can be formulated from a mixture of mineral oil, self-emulsifyingbeeswax and water in which mixture the active ingredient, dissolved in asmall amount of an oil such as almond oil, is admixed. A typical exampleof such a cream is one which includes about 40 parts water, 20 partsbeeswax, 39 parts mineral oil and about 1 part almond oil.

Ointments may be formulated by mixing a solution of the activeingredient(s) in a vegetable oil such as almond oil with warm softparaffin and allowing the mixture to cool. A typical example of such anointment is one which includes about 30% almond oil, and about 70% whitesoft paraffin by weight.

Lotions may be conveniently prepared by dissolving the activeingredient, in a suitable high molecular weight alcohol, such aspropylene glycol or polyethylene glycol, having a molecular weight ofabout 300 to about 1450 MW.

It should be recognized that misoprostol will break down toprostaglandin A if in the presence of water. Thus, creams lastapproximately only one week because of this stability problem.Therefore, misoprostol is best used in a powder form that can behydrated at the time of use. Misoprostol is more stable in a vehiclethat uses propylene glycol. It can be stored in a dry state for a coupleyears if it is stabilized with hydroxypropyl methyl cellulose. In liquidform as the oil, it is preferably dissolved in absolute alcohol(ethanol) and frozen. The frozen alcohol and misoprostol mixture isstable for at least a year.

In addition, plasticizers, additives, colorants, preservants, andprotectants may be added to the present pharmaceutical compositions toenhance aesthetic and mechanical features (i.e. softness andflexibility). The type and amount of plasticizer, additive, colorant,preservant, and/or protectant to be used can be readily optimized toachieve a desired effect. Also, surfactants and drying agents may beadded as well to the pharmaceutical composition to achieve a desiredeffect. Non-limiting examples of surfactants include stearoyl lactylate,calcium steroyl lactylate, and glyceryl monosterarate. Non-limitingexamples of drying agents include magnesium aluminum silicate andsodium, magnesium and potassium sulfate, and hydrophilic clays. Thepresent pharmaceutical compositions may also contain taste modifiers,coloring agents, and moisture retaining agent. Examples of tastemodifiers include non-reducing sugars, such as xylitol, malitol, orLycasin® manufactured by Roquette America Inc. of Keokuk, Iowa. Examplesof moisture retaining agents include celluloses, cellulose derivatives,starches, starch derivatives, vegetable gums, non-hygroscopic mono- anddi-oligosaccharides, and silicon dioxide.

One or more additional substances which have therapeutic effects on theskin and/or tissue being treated may also be incorporated in thecomposition. For example, other suitable types of active ingredients maybe incorporated in the compositions of this invention and may includeother compounds known to have beneficial effect on skin and/orcollagen-containing tissue. Additionally, the pharmaceutical compositionof the present invention may optionally include an anti-inflammatoryagents and/or a local anesthetic, such as dyclonine in an amount ofabout 0.5 to 1% by weight.

Dosage formulations of the present pharmaceutical compositions can beprepared by combining them with a pharmaceutically acceptable carrier,such as a slow release agent, to make either immediate or slow releaseformulations as is well known in the art. Such pharmaceuticallyacceptable carriers may be either solid or liquid in form such as, forexample, cornstarch, lactose, sucrose, peanut oil, olive oil, sesameoil, propylene glycol and water. If a solid carrier is used, the dosageformulation of the present pharmaceutical compositions may be in, forexample, powder, troche, or lozenges form. If a liquid carrier is used,the dosage formulation of the present pharmaceutical compositions may bein, for example, soft gelatin capsule, syrup liquid suspension,emulsion, or solution form. The dosage formulations may also containadjuvants, such as preserving, stabilizing, wetting or emulsifyingagents, or solution promoters. Immediate and slow release formulationsare well known in the art and have been described, for example, in U.S.Pat. No. 4,764,377 (the disclosure of which is incorporated herein byreference), which describes a method for treating periodontal disease bymeans of a delivery device placed within the periodontal pocket so thatrelease of an antibiotic, such as tetracycline, occurs in the immediatevicinity of the disease process.

In the method of the present invention, the pharmaceutical compositionsare administered to a patient one or more times daily. For example, thepresent pharmaceutical composition is typically administered from aboutone time to about six times daily, more typically about two times toabout five times daily, and most typically about three times to aboutfour times daily.

As presented herein, methods of the instant invention for treatingperiodontal disease can optionally further comprise administering to apatient at least one step of sub-gingival scaling, root planning, orcurettage. These procedures involve manually removing calculus, plaqueand other deposits, smoothing the root surface to rid of necrotic toothsubstances, and curetting the inner surface of the gingival wall of theperiodontal pockets to separate away the diseased soft tissue.

In the method of the present invention, administering a pharmaceuticalcomposition containing misoprostol, in combination with an antibiotic,was found to be unexpectedly superior to administering eithermisoprostol or antibiotic alone, or the sum of their individualcontributions. Amelioration of periodontal disease by the presentpharmaceutical compositions is indicated by at least one of thefollowing characteristics: regeneration of tooth-supporting tissues,improvement of periodontal-involved teeth, regeneration of infra bonydefects, regeneration of alveolar bone or tissue, reduction of pocketdepths, decrease in tooth mobility, reduction in soft tissueinflammation or edema, or reduction in bacterial colonization orinfection. Topical application of misoprostol with doxycycline, forexample, resulted in statistically significant improvements in pocketdepth when compared to treatment with doxycycline or misoprostol alone,or the sum of their individual contributions. The results of this studyindicate that topical administration of misoprostol, with and withoutpre-treatment with doxycycline, results in significant improvements inperiodontal disease compared to the current standard of care,doxycycline and/or sub-gingival planing, scaling and curettage. Althoughthere were no statistically significant differences between the threeactive treatments at six weeks post-treatment, at 52 weekspost-treatment, administration of doxycycline+misoprostol andplacebo+misoprostol resulted in statistically significantly greaterimprovements in mean pocket depths compared to doxycycline+placebo. Infact, most patients in the doxycycline+placebo group showed regressionof the disease at 52 weeks post-treatment, where changes in pocketdepths in the doxycycline+placebo group were statisticallyindistinguishable from those in the placebo group. See FIGS. 1-4.

Effect of Scaling, Root Planing and Curettage The scaling, rootplanning, and curettage procedure, even without additional treatment,benefits the patient by providing an approximately 25-30% reduction inpocket depth as long as one year later. However, there is a continuingsearch to find methods of increasing the level of benefit, andprolonging the period of effectiveness.

Effect of Doxycycline Doxycycline is a tetracycline antibiotic withactivity against anaerobic bacteria. A 10-day pre-treatment program isintended to significantly reduce the bacterial colonization in theinterface between the soft tissue and hard tissue structures. A full10-day period may not be necessary to have produced just this effect,but the additional time also allows the infection-induced inflammationand edema in the soft tissues to subside and be considerably reduced.The subsequent scaling and root planing then removes most or all of thecalculus and biofilm that promote the bacterial colonization.

Doxycycline treatment, or any other antibiotic treatment by itself, isunlikely to be consistently successful in a large proportion of thepatients with moderate or severe periodontal disease of the type treatedin this study. First, removal of the calculus and destruction of thebiofilm along with removal of chronic tissue that lines the diseasedpocket are critical for the elimination of sites that serve asbioreservoirs for reinfection and which promote recolonization. Second,the effectiveness of all commercially available antibiotics can becircumvented by the emergence of resistant strains. Low antibioticconcentrations and long-term, chronic treatment are well known to serveas environmental pressures that can lead to the emergence of resistantstrains. The emergence of a resistant strain will very probably becharacterized by a recurrence of the infection, inflammation, edema andtissue sensitivity that characterized the original disease. Third, whilethis study employed oral doxycycline as the antibacterial medication,there are other commercially available products that directly target thegingival tissue by slow release of antibiotic from string, wafer, or gelembedded in the pocket between the soft and hard tissues. These productshave the disadvantage of physically separating the two tissues, thusinhibiting the attachment of the two structures, and ultimately, closureof the pocket.

As expected with any antibiotic, halting the administration of the drug,without controlling the sources of reinfection, ultimately leads to aresurgence of the disease state, and a return to the previous state ofinfection or even further progression of the disease. Patientsrandomized to the doxycycline pre-treatment program alone showed thispattern of response. While there was an early positive response that wasat least as good as any of the other options, the initial benefitdissipated completely within one year so that at the end of that yearthe treatment program was indistinguishable from a placebo treatmentwith scaling and root planing, no matter how deep or shallow theoriginal pocket depth.

Effect of Misoprostol Misoprostol is a prostaglandin E1 analog that hasbeen implicated in the stimulation of bone growth and remodeling and inthe proliferation of connective and epithelial tissues. (Marks, S. C.Jr., Miller, S. C. Local infusion of prostaglandin E-1 stimulatesmandibular bone formation in vivo. J. Oral Pathol 1988, 17: 500-505;Marks, S. C. Jr., Miller, S. C. Site-directed formation of new lamellarbone in adult dogs by infusion of prostaglandin E-1, Dixon, A. D.,Samat, B. G., Hoyte, D. A. N., eds. Fundamentals of bone growth:Methodology and applications. Boca Raton, Fla.: CRC Press: 1991:375-381; Marks, S. C. Jr., Larson, E. K., Bowman, B. M., Miller, S. C.Local induction of alveolar bone in adult dogs by infusion ofprostaglandin E-1, Davidovitch, Z, ed., Biological mechanisms of toothmovement and craniofacial adaptation. Columbus, Ohio: Ohio StateUniversity Press: 1992, 137-142; Marks, S. C. Jr., Miller, S. C. Localdelivery of prostaglandin E-1 induces periodontal regeneration in adultdogs. J. Periodont Res. 1994, 29: 103-108; and Flanagan, A. M.,Chambers, T. J. Stimulation of bone nodule formation in vitro byprostaglandin E-1 and E-2. Endocronology 1992, 130: 433.) Misoprostol'srole as a commercial product (CYTOTEC®) as a cytoprotective agent may bedue, at least in part, to this stimulatory proliferative effect onepithelial cells in the gut. Local administration of misoprostol as partof these dental procedures was suggested by results published by othersthat showed prolonged, low dose administration of prostaglandinsstimulated growth of alveolar bone, connective tissue fibers forreattachment and new cementum. The new alveolar bone was laid downwithout previous bone resorption.

Local application of the misoprostol-containing gel by the patients inthis study enhanced the beneficial effect of scaling, root planning andcurettage in the short-term, and added to and prolonged the enhancedbenefits provided by doxycycline pre-treatment. Since misoprostol'spostulated mechanism of action in this setting, promoting bone growth isnot a rapid process and takes substantial time to show its cumulativeeffect, it is not surprising that significant added benefit was not seenby the Week 6 observation. However, the benefit continued to accruebeyond that time point, even though the administration of misoprostolwas no longer continuing.

This continued accrual of effect is not necessarily an indication of a“prolonged duration of action” for misoprostol, but is probably just theresult of misoprostol stimulating the development of some vital, butunmeasured, tissue infrastructure. For instance, it is possible that the6 weeks of misoprostol application stimulated the formation of a bonelattice structure in the region cleaned of calculus, biofilm and chronictissue by the scaling, root planning and curettage. This would not besubstantial enough at 6 weeks to resist probing, but would be sufficientto promote the activity of osteoblasts over the intervening severalmonths. Freeing the surfaces and tissue of bacterial colonization by thedoxycycline pre-treatment would make this process even more likely tosucceed, and promote the involvement of more surface area. Consistentwith this hypothesis is the observations of the principal investigatorat the quarterly return visits that patients within the treatment groupsultimately identified as receiving misoprostol, showed improvements intooth mobility, whereas patients in the other groups did not. This wouldbe in spite of the fact that pocket depths and improvements in pocketdepth were apparent in all cases. Improvements in pocket depth requiresonly the close adhesion of the epithelial cells to the hard tissues,whereas decreased tooth mobility requires laying done of additional bonematrix.

Radiographs should be able to be used to document the formation of newbone within the previously diseased areas. Unfortunately in this study,the attempt to provide this information was hindered by the unexpecteddeterioration of the impression materials that were used to constructthe step-wedges to index the “before” and “after” radiographs. While itis possible to detect apparent increases in bone tissue near the rootsof the teeth, the quantitative interpretation is open to the criticismthat the results might be biased by alterations in the angle of viewing.

Another conclusion provided by these results is that more severeperiodontal disease of this type may be able to be treated by successiverounds of scaling, root planning and curettage with doxycyclinepre-treatment and misoprostol post-treatment. It might well be possibleto completely fill in even very large pockets if one successivelyrepeats the treatment program, tailors the treatment repeat scheduleover a period of time that allows the bone to be built up in successivelayers.

Summary This data provided herein is based in part on a Phase IIexploratory study examining the efficacy of misoprostol in the treatmentof moderate-to-advanced periodontitis. These results indicate thatmisoprostol provides an important non-invasive step in the algorithm totreat these patients. None of the patients who received pretreatmentwith doxycycline followed by topical application of misoprostol alonefor 6 weeks showed regression of the initial improvements one full yearafter treatment. Importantly, none of the patients who receivedmisoprostol required surgical intervention to stop the progression ofthe disease. Finally, it was unexpectedly discovered that an evengreater decrease in pocket depth and a decrease in teeth mobility wasobtained when the mammalian (human) patients were pretreated with asystemic regimen of an antibiotic having activity against plaqueproducing bacteria and subsequently treated for three weeks with apharmaceutical composition comprising both misoprostol and an antibiotictopically administered to, at, on, onto, in, into or near theperiodontal pocket. See Table 6. The ability to reduce the treatmentperiod from six (6) weeks to three (3) weeks is significant because notonly did it provide superior results but it also insured a greaterlikelihood that patients would comply with the treatment regimen so asto receive the full benefit of the treatment.

Tomar, S. L., Asma S. Smoking-Attributable Periodontitis in the UnitedStates: Findings From NHANES III. J Periodontol 2000, 71: 743-751 hasestimated that 9.2% of dentate adults develop periodontitis (pocketdepths ≧4 mm), which projects to about 15 million cases of periodontitisamong U.S. adults. With these large numbers of adult patients presentingfor treatment, the addition of effective non-surgical interventionsusing oral medications would be advantageous based on ease ofadministration, minimal invasiveness and cost. In our opinion, thecombination of misoprostol and an appropriate antibiotic has tremendouspotential in the treatment of periodontal disease.

Overall Conclusions Scaling, root planning and curettage provided anapproximately 25%-30% reduction in pocket depth after 6 weeks, and thatwas maintained for at least one year.

Topical applications of misoprostol (12 μg/site QID) for 6-7 weeks incombination with doxycycline pre-treatment (100 mg BID/QD for 10 days)resulted in statistically significant improvements in pocket depths at52 weeks post-treatment compared to doxycycline pre-treatment alone inpatients with moderate-to-advanced periodontitis.

Topical applications of misoprostol (12 μg/site QID) for 6-7 weeks, withand without doxycycline pre-treatment (100 mg BID/QD for 10 days),resulted in statistically significant improvements in pocket depths at52 weeks post-treatment compared to the standard treatment in the art(i.e., scaling, root planning and curettage) in patients withmoderate-to-advanced periodontitis.

At 52 weeks post-treatment, the improvements in pocket depths followingpre-treatment with doxycycline (100 mg BID/QD for 10 days) followed bypost-treatment with misoprostol (12 μg/site QID) for 6-7 weeks werenumerically greater than improvements following the standard treatmentin the art (i.e., scaling, root planning and curettage) followed bypost-treatment with misoprostol (12 μg/site QID for 6-7 weeks) alone.

Doxycycline pre-treatment alone prior to the scaling, root planning andcurettage procedure provided a short-term enhancement of reduction inpocket depth. Most of the effect can be ascribed to reduction in softtissue inflammation and edema associated with reduction in the bacterialcolonization and infection. However, doxycycline pre-treatment prior tothe scaling, root planning and curettage procedure failed to provide anyadditional benefit at 52 weeks post-treatment beyond that provided bythe scaling, root planning and curettage alone.

Misoprostol, applied locally in low doses (12 μg/site QID) for 6-7weeks, resulted in the enhancement of the benefits of scaling, rootplanning and curettage. Initial effects in reducing pocket depth aredetectable by 6 weeks, especially for deeper pockets and persist for 52weeks.

The combination of systemic doxycycline pre-treatment and topicalmisoprostol post-treatment for 6-7 weeks resulted in recovery patterncharacterized by a rapid onset (i.e., detectable at 6 weeks) andcontinued improvement over the long term (i.e., 52 weeks).

Misoprostol post-treatment, but not doxycycline pre-treatment, ischaracterized by decreases in tooth mobility, along with the decreasesin pocket depth.

Unexpectedly superior results were obtained by systemically pretreatingthe patient with an antibiotic, such as doxycycline (100 mg BID/QD for10 days), prior to topically administering misoprostol for 6 weeks tothe periodontal pocket. Even more exceptional results were obtained in ashorter time (3 weeks) when the topically administered misoprostol wasco-administered with a topically administered antibiotic, particularlyin the same topical formulation. As reflected in Table 6, the meanchange (decrease) in pocket depth for patients presenting baselinepocket depths of 15 mm was a 7.7 mm decrease for patients pretreatedsystemically with an antibiotic and topically treated with misoprostolfor 6 weeks, or a 9.13 mm decrease for patients pretreated systemicallywith an antibiotic and topically treated with misoprostol and anantibiotic alone for only 3 weeks. Similar results appear in Table 6 forother baseline pocket depths as well. For example, the mean change(decrease) in pocket depth for patients presenting baseline pocketdepths of 9 mm was a 4.04 mm decrease for patients pretreatedsystemically with an antibiotic and topically treated with misoprostolfor 6 weeks, or a 4.96 mm decrease for patients pretreated systemicallywith an antibiotic and topically treated with misoprostol and anantibiotic alone for only 3 weeks.

The following examples illustrate the unexpectedly superior efficacy ofthe present pharmaceutical composition and method of treatingperiodontal disease with said pharmaceutical compositions in accordancewith the instant invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples are provided for illustrative purposes and arenot to be construed to limit the scope of the claims in any mannerwhatsoever.

Example 1 Comparative Study on Effects of Administering Misoprostol orDoxycycline Alone or in Combination

1.1 Overall Study Design and Plan This was a randomized, double-blind,single-center, multiple-dose, parallel-group, placebo-controlled study.At baseline, patients presenting at Periodontal Specialists of Ames forthe treatment of periodontitis were screened for the study. Patientswere assessed using medical histories, dental histories, blood pressure,pulse, pregnancy tests (if female), periapical radiographs andperiodontal examinations. Patients who met all of the inclusion andexclusion criteria, appeared in good health other than periodontitis,and who had documented moderate-to-advanced periodontitis with at leastfour destructive periodontal-diseased pockets (ranging from 5 to 12 mmin depth) were eligible for study participation. Each patient underwenta gross debridement, following which, he/she was randomized to one offour treatment groups:

Treatment Group Assigned Treatment A Placebo B Doxycycline 100 mg BID/QDfor ten days pre-treatment C Doxycycline 100 mg BID/QD for ten dayspre-treatment + Misoprostol 10 ug/site QID for six weeks post-treatmentD Misoprostol 10 ug/site QID for six weeks post-treatment

Patients randomized to receive doxycycline (Groups B and C) wereadministered a loading dose of doxycycline 100 mg BID for one day andthen instructed to take doxycycline 100 mg QD for nine consecutive days;patients in Groups A and D received matching doxycycline placebo for tendays.

On the last day of doxycycline/placebo administration, patients reportedto the clinical research facility and underwent (under local anesthesia)dental sub-gingival scaling, root planing, and curettage on one side ofthe mouth. Following completion of these procedures, patients randomizedto misoprostol (Groups C and D) received an application of a misoprostolpaste (10 ug/site) and those randomized to placebo (Groups A and B)received placebo paste. Patients were instructed to apply the paste toeach quadrant four times daily for the next week. One week later,patients returned to the clinical research facility and underwentsub-gingival scaling, root planing, and curettage on the other side ofthe mouth. The misoprostol/placebo paste was administered as describedabove. Patients were instructed to apply the paste to the affected teethfour times daily for an additional six weeks. They were also instructedto the proper use of the rubber tip for six weeks.

At two weeks post-treatment, patients were contacted for adverse eventmonitoring and compliance with study drug administration. Six weeksfollowing the second sub-gingival scaling, planing, and curettageprocedure, patients returned to the clinical research facility forperiodontal and adverse event assessments. Dental prophylaxis, includingperiodontal maintenance was repeated at the three month intervals, withfinal assessments performed at 52 weeks post-treatment.

1.2 Discussion of Study Design, Including Choice of Control GroupsPrevious experience using misoprostol with periodontal regeneration inthis facility has indicated a greater response when doxycycline wasadministered prior to scaling. Therefore, treatment arms usingdoxycycline alone, misoprostol alone, and misoprostol and doxycyclinewere included to investigate the synergistic effect of these drugs. Theuse of placebo did not endanger the patients and is considered standardin an efficacy trial. The treatment schedule is also considered standardpractice for the treatment of periodontitis.

1.3 Study Population A total of 52 patients (13 per treatment group)were to be enrolled into the study. Patients meeting all of theinclusion criteria and none of the exclusion criteria were eligible forstudy participation.

1.3.1. Inclusion Criteria Patients were to meet all of the followinginclusion criteria to enter the study:

-   -   1. Signed the basic consent form after reading the information        summary.    -   2. Moderate to advanced localized chronic adult periodontitis,        at least 20 natural teeth, and at least four periodontal pockets        with probing pocket depths between 5 mm and 12 mm. There should        not have been periodontal pockets where the depth of the pocket        corresponded to the apex of the tooth as it might have developed        an endodontic/periodontic combined lesion.    -   3. Evidence of crestal interdental bone loss in baseline        periapical radiographs.    -   4. Class III or Class IV periodontal diagnosis, as determined by        the standards of the American Academy of Periodontology.    -   5. Age between 19 and 75 years and in good systemic health as        determined by a complete medical history, blood pressure and        pulse rate measurements.    -   6. Ability to follow instructions for dental hygiene and ability        to return for specified examinations.

1.3.2. Exclusion Criteria Patients were to be excluded from the study ifany of the following conditions were present:

-   -   1. Periodontitis treatment within six months prior to the        baseline exam.    -   2. Compromised heart condition that required antibiotic        prophylaxis for prevention of infective endocarditis during        dental procedures.    -   3. Joint replacement prostheses requiring antibiotic        prophylaxis.    -   4. Previous or concurrent cancer treatment.    -   5. Evidence of compromised kidney function in medical history    -   6. Evidence of acute or chronic systemic infection such as        herpes simplex, tuberculosis, AIDS in medical history.    -   7. Evidence of clinically significant chronic systemic disease        such as cardiovascular disease, uncontrolled diabetes mellitus,        hepatitis, lupus in medical history.    -   8. History of, or current clinical signs of, oral candidiasis.    -   9. Allergies to ingredients within the test and/or placebo        agents.    -   10. Medication use that could have caused gingival enlargement,        such as dilantin, cyclosporin, nifedipine, etc.    -   11. Antibiotic use within the past six months or concurrent        antibiotic use.    -   12. Current non-steroidal anti-inflammatory drug use, or within        one month of baseline exam.    -   13. Use of dentrifirice or rinses containing chlorhexidine or        sanguinaria within one month of baseline exam.    -   14. Receipt any other investigational drug or device.    -   15. In the examiner's opinion, inability to comply with the        instructions of the study.    -   16. Evidence of pulpitis.    -   17. Patients with bruxism who could not/would not wear a mouth        guard.    -   18. Current use of tobacco products.    -   19. Pregnant women, women with a positive serum pregnancy test        completed 10 days prior to study entrance, and childbearing age        women not using non-hormonal (barrier) methods of contraception.    -   20. Acute periodontal infections.

1.3.3 Removal of Patients from Therapy or Assessment Patients were to bewithdrawn from the study if a particular site lost more than 2 mm ofattachment at any of the post-treatment examinations. Patients were alsoto be withdrawn if the plaque index, a measure of post-treatmentprotocol noncompliance, had a score of >0 after the screening exam.

If an individual tooth developed pulpitis post-treatment, the data forthat tooth were excluded from the analysis. The patient was allowed toremain in the study, and all data for the remaining evaluable teeth wereused in the analysis.

1.4 Treatments

1.4.1 Treatments Administered

Patients were randomized to one of four treatments:

Treatment Treatment Group Study Medications Administered GroupAssignment Pre-treatment Post-treatment A Placebo Placebo BID for 1 day,Placebo Paste followed by QD for 9 days QID for 6-7 weeks B DoxycyclineDoxycycline 100 mg BID for 1 day, Placebo Paste followed by 100 mg QDfor 9 days QID for 6-7 weeks C Doxycycline + Doxycycline 100 mg BID for1 day, Misprostol paste Misoprostol followed by 100 mg QD for 9 days 10ug/site QID for 6-7 weeks D Misoprostol Placebo BID for 1 day,Misprostol paste followed by QD for 9 days 10 ug/site QID for 6-7 weeks

Doxycycline and doxycycline placebo were administered orally. Themisoprostol and placebo pastes were administered topically.

1.4.2 The Pharmaceutical Agents Commercially-produced doxycycline 100 mgcapsules (lot number KH7-073—Medisca), and commercially-producedmisoprostol 200 μg tablets (lot numbers 68N643, 68H623, 8P649, 9M671,9H675—Searle) were obtained from their respective manufacturers.

Doxycyline was overencapsulated in #3 yellow/gray gelatin capsules (lotnumber 62371) in order to maintain the study blind. Empty capsules wereused for the doxycycline placebo.

The misoprostol and placebo pastes were prepared by a pharmacist. Themisoprostol tablets were ground to a fine powder and prepared asdescribed below.

For the misoprostol paste, misoprostol was mixed with polyethyleneglycol flakes (carbowax), polyethylene glycol, SUPER WERNET'S® DentureAdhesive Powder and absolute ethanol to a final concentration of 50ug/ml. The mixture was loaded into 10 ml syringes by the staffpharmacist; this provided a total of 50 application sites per syringe.

For the placebo paste, the mixture of polyethylene glycol flakes(carbowax), polyethylene glycol, SUPER WERNET'S® Denture Adhesive Powderand absolute ethanol was loaded into 10 ml syringes by the staffpharmacist; this provided a total of 50 application sites per syringe.

1.4.3 Method of Assigning Patients to Treatment Patients were randomlyassigned to each treatment by the University of Iowa Dental School staffpharmacist, Karen Baker. The pharmacist assigned each patient a numberbetween 1 and 72 and labeled all study medications for that patient withthe appropriate number.

1.4.4 Selection of Doses in the Study The doxycycline regimen use inthis study is considered standard practice for periodontitis treatment.The misoprostol dose used in the study was based on the Investigator'sprevious experience in the clinic with treatment of aphthous ulcers andlichen planus with misoprostol.

1.4.5 Timing of Dose for Each Patient Doxycycline and matching placebowere administered orally on a daily basis for ten days, without regardto standard water or food intake. Patients were instructed to takemedication at approximately the same time each day.

The initial dose of the misoprostol/placebo paste was applied by theperiodontist, and all subsequent doses were applied by the patient. Eachaffected tooth was wiped with a 2×2 sponge to dry the tissue. Then 0.2ml of the paste was dispensed from the 10.0 ml syringe onto the tip ofthe finger and applied to each affected tooth. The patient then sipped alittle water and gently flushed the paste to gel it. Patients wereinstructed not to eat anything for at least one hour.

1.4.6 Blinding This was a double-blind study. All study medications wereprepared and labeled with a number code; this code, which correspondedto the patient's assigned treatment, was not known to the Applicants,any other study site personnel, or study patients. Overencapsulation wasused to maintain the blind for doxycycline.

1.4.7 Prior and Concomitant Therapy Concomitant medications were allowedduring the study. Patients were encouraged not to change the dosageregimen during the 7-week treatment period. Concomitant medication useduring the 52-week follow-up period was not monitored.

1.4.8 Treatment Compliance Treatment compliance was assessed byquestioning the patient about the application of the study medication.Quantitative assessments of treatment compliance were not performed.

1.5 Efficacy and Safety Variables

1.5.1 Efficacy and Safety Measurements Assessed Efficacy was assessedusing periodontal examinations and periapical radiographs. Safety wasassessed primarily through adverse event monitoring. The schedule ofobservations and procedures is presented in Table 1.

TABLE 1 Schedule of Observations and Procedures Baseline- 6 Wk 12 MoProcedure 10 days Day 1 1 Wk 2 Wk (a) 3 Mo 6 Mo 9 Mo (b) Medical HistoryX Dental History X Blood Pressure, Pulse X X X X X X X X X AAPClassification X Pregnancy Test X Periodontal Exam Pocket Depth X X X XX X Bleeding X X X X X X Attachment Level X X X X X X Plaque Index X XPeriapical Radiograph X X X Gross Debridement X X X X X Sub-GingivalScaling X X (c) X (c) X X X X and Planing Doxycycline/Placebo X XAdministration (d) Misprostol/Placebo X ——————————

 X Administration (e) Adverse Event X X X X X X X X Monitoring (a)Relative to second scaling and planing procedure. (b) Study blind wasbroken at 52 weeks. Patients who had not received misoprostol wereoffered the option to enroll in a six-week open-label extension in whichthey received misoprostol for six weeks. (c) Procedure performed on onehalf of the mouth on Day 1 and the other half of the mouth one weeklater. (d) Initiation of doxycycline for 10 days prior toscaling/planing on Day 1; last dose completed on Day 1. (e) Administeredfour times daily for a total of 6 weeks on one side of the mouth and 7weeks on the other side.

1.5.1.1. Periapical Radiographs Four periapical radiographs usingstepwedges were performed at baseline, 6 weeks and 52 weeks. Stepwedgeswere used to maintain accuracy at recall intervals. Kodak Ultra-speed(D-speed) film was used (Eastman Kodak Co., Rochester N.Y.). Filmexposures were made under standard setting of 70 kVp, 15 mA, 16 inchfixed focal image distance (FID). These exposure settings weremaintained throughout the course of the study. All dental radiographswere processed manually using freshly mixed Kodak GBX developer andfixer with strict adherence to the manufacturer's guidelines with regardto time and temperature control. All conventional radiographs wereilluminated and digitized using an Imapro QCS 3200 flat bed scanner(Imapro Corp, Ogdensburg N.Y.) with a pixel spatial resolution of 0.042mm and an 8-bit gray scale depth. The QCS 3200 provided a lamp drift ofless than 2% over a scan. All radiographic images were archived onoptical disks. Crestal bone height was measured using periapicalradiographs for posterior teeth and anterior teeth. At least four sitesshowing pocket depth of 5-12 mm were selected for each patient. Forthese x-rays, stepwedges were used for contrast and density correction.Two radiographic images for each anatomic site was projected on thecomputer monitor when making crestal bone height measurements. This“side-by-side” image projection provided greater consistency in theidentification of reference landmarks.

In order to assess any changes in alveolar crestal height,cemento-enamel junction (CEJ) to alveolar crest distance measurement wasmade mesially and distally, parallel to the long axis for each tooth oneach set of two images.

Radiographs were digitally enhanced using the Adobe Photoshop AdjustLevels tool. This allowed the digital images to all have roughly thesame range of contrast and brightness. Performing just the normalizationimproved the qualitative comparison of the images by standardizing (fromimage to image) the gray scales that represent the different tissuetypes.

Once normalized, additional enhancements of the images were performed.One simple technique was to assign colors to the gray scales (pseudocoloring). This technique takes advantage of the fact that our vision ismore sensitive to changes in color than in changes in gray scale. Thesepseudo colored images make it much easier to discriminate differencesbetween radiographs.

Another enhancement technique was to combine a threshold, inverted imagewith the normalized image. This was done using a multiplicationoperation in the layers palette of Photoshop. This operation enhancedthe region of the treatment area.

Bone density was estimated using semi-quantitative techniques where grayscale values in the digital images were calibrated to a rough bonedensity scale. To do this, a region in a bone-(or tissue)-free region ofthe radiograph was defined and it's mean gray scale was defined as thezero-density point. Then a region in a carie or other bright point inthe radiograph was defined and it's mean gray scale was defined as the100% dense point. Using these two points a scale linking gray scalemeasurements to bone density was constructed and applied to measurementsof regions defined in the treatment area.

These bone density measurements and pseudo coloring were done with ImageJ a program developed by Wayne Rasband at the Research Services Branch,National Institute of Mental Health, Bethesda, Md. Adobe Photoshopversion 6.0 was used for normalization and gray scale operations.

1.5.1.2. Periodontal Examination by

1.5.1.2.1. Periodontal Pocket Probing Depth Periodontal probing depthswere measured at baseline, at six weeks post-treatment, and at 3, 6, 9and 12 months post-treatment. More recently, a larger study at threeweeks post treatment was performed as described in Example

To measure the periodontal pocket probing depth, a manual University ofMichigan off-set probe marked in millimeters was inserted into thepocket with the long axis parallel to the long axis of the tooth.Insertion was continued to the base of the pocket with gentle pressureuntil a distinct resistance was felt.

The measurement from the tip of the probe to the gingival margin (GM-BP,gingival margin to the base of the pocket) was recorded in millimeters.On measurements of proximal sites the gingival margin may cross theprobe at an angle, and the highest point on the side of the probe wasread. If the distance fell between two probe graduations, the number wasrounded to the nearest whole numbers. Pocket measurements were recordedat the following six sites around the tooth:

Mesio-buccal with side of probe touching both teeth in embrasure nearthe contact point. If there was no adjacent tooth, the probe must havebeen aligned at the line angle of the tooth. Mid-buccal If the tooth wasrotated, measurements were made in the middle of the exposed surface andnot at the anatomical middle of the tooth. Disto-buccal with the side ofthe probe touching both teeth in the embrasure near the contact point.If there was not adjacent tooth the probe was to be aligned at the lineangle of the tooth. Mesio-lingual with the side of the probe touchingboth teeth in the embrasure near the contact point. If there was noadjacent tooth, the probe was to be aligned at the lie angel of thetooth. Mid-lingual if the tooth was rotated, the measurement was done inthe middle of surface and not at the middle of the anatomic mid-lingual.Disto-lingual with the side of probe touching both teeth in theembrasure near the contact point. If there was no adjacent tooth, theprobe was to be aligned at the line angle of the tooth.

In measuring the pockets, the long axis of the probe was kept asparallel to the long axis of the tooth as possible.

1.5.1.2.2. Bleeding Upon Probing Bleeding upon probing was assessed atbaseline, at six weeks post-treatment, at 3, 6, and 9 monthspost-treatment, and at 52 weeks (12 months) post-treatment. Afterremoving the probe from a pocket, the site was observed and scored from0-3 as follows:

0 No Bleeding 1 A small point of blood seen at the probe exit site, or avery thin line of blood 2 Bleeding that filled the interdental embrasureor ran along margin as more than a thin line 3 Profuse bleeding seenimmediately after probe removal

1.5.1.2.2. Probing Attachment Level Attachment levels were measured atbaseline, at six weeks post-treatment, at 3, 6, and 9 monthspost-treatment, and at 52 weeks (12 months) post-treatment. To measurethe probing attachment level at fixed reference was used as a landmark.The cemento-enamel junction (CEJ) was located by placing the periodontalprobe against the enamel surface coronally to the gingival margin andwith the probe at about 45 degrees to the long axis, the tip was movedaxially along the enamel until a change in direction of “bump” was feltat the CEJ.

If the gingival margin was on enamel, the measurement from the probe tipto the GM (GM-CEJ) was recorded as a positive number.

If the gingival margin was on the root, the measurement from the CEJ tothe gingival margin was recorded as a negative number. The GM-CEJ wassubtracted from the GM-BP to determine the probing attachment level. Ifthe CEJ was not clearly detectable, then the incisal edge (Landmark X)or a restoration margin (landmark R) may have been used. Landmark X datawere negative numbers. Landmark R data were negative numbers if therestoration margin was coronal to the gingival margin. It was better touse an R landmark than an uncertain CEJ.

5.1.2.3. Plaque Index Plaque index was measured and graded at baselineand six weeks post-treatment using the following criteria:

0 No plaque in the gingival area 1 A film adhering to the free gingivalmargin and adjacent area of the tooth 2 Moderate accumulation of softdeposits within the gingival pocket on the gingival margin and/oradjacent tooth surface that could be seen by the naked eye 3 Abundanceof soft matter within the gingival pocket and/or on the gingival marginand adjacent tooth surface

1.5.1.3. American Association of Periodontology Classification Based onperiodontal examinations and periapical radiographs, only patients withClass III or Class IV periodontal classifications were eligible forenrollment in the present study.

Class Type Symptoms I Gingi- No attachment loss vitis Bleeding may ormay not be present Pseudopockets may be present Only the gingivaltissues have been affected by the inflammatory process II Early Bleedingupon probing may be present in the Peri- active phase odontitis Pocketdepths or attachment loss of 3 to 4 mm Localized areas of recessionPossible Class I furcation invasion areas Horizontal type of bone lossis most common Slight loss of the interdental septum Alveolar bone levelis 3 to 4 mm from the CEJ area III Moderate Pocket depths or attachmentloss of 4 to 6 mm Peri- Bleeding upon probing odontitis Grade I and/orGrade II furcation invasion areas Tooth Mobility of Class I Horizontalor Vertical bone loss may be present Alveolar bone level is 4 to 6 mmfrom the CEJ area Radiographic furcations of Grade I and/or Grade IICrown to root ratio is 1:1 (loss of ⅓ of supporting alveolar bone) IVAdvanced Bleeding upon probing Peri- Pocket depths or attachment lossover 6 mm odontitis Mobility of Class II or Class III Horizontal andvertical bone loss Alveolar bone level is 6 mm or more from the CEJ areaRadiographic furcations Crown to root ratio is 2:1 or more (loss of over⅓ of the supporting alveolar bone)

1.5.1.4. Adverse Events Patients were queried for adverse events at allstudy visits. In addition, patients were instructed to call the studysite to report any adverse side effects of the study medication.

1.5.1.5. Vital Sign Measurements Blood pressure and pulse measurementswere taken at all study visits to ensure patients were in good health.These data were not entered into the database.

1.5.1.6. Appropriateness of Measurements The measurements performed inthis study are considered standard practice for the treatment ofperiodontitis.

1.6 Data Quality Assurance To ensure consistent measurements, allperiodontal assessments were performed by the Principal Investigator.Data were entered into an Excel database and a 100% audit of the pocketdepth data was performed.

1.7 Statistical Methods and Determination of Sample Size

1.7.1 Statistical and Analytical Methods All analyses are performedusing the NPAR1WAY or TTEST procedures in SAS. Plots are created usingMINITAB.

1.7.1.1 Efficacy Analyses Mean changes in pocket depth were determinedat 6 and 52 weeks post-treatment. Patients presented with baselinepocket depths of ≦4, 5, 6, 7, 8, 9, 10, 12, and 15 millimeters. Separateanalyses were performed for each baseline pocket depth between ≦4 and 10mm; however, due to small sample sizes, analyses were not performed forthe remaining baseline pocket depths. Mean changes in pocket depths werecalculated per patient and per measurement. Standard deviations forpatient means were presented, however, since a positive correlation wasexpected between the change in pocket depth for the same patient,standard deviations were not reported for these measurements. In orderto evaluate the normality of the distribution of the patient means,skewness and kurtosis values, which are equal to 0 for a normaldistribution, were presented.

Pairwise comparisons were performed using the nonparametric Wilcoxonrank-sum test. The test assumes that either the two treatmentdistributions are equal or one distribution is stochastically largerthan the other. These assumptions are much weaker than the assumption ofnormal distributions with equal variance. Dot plots for each sample wereinspected for any serious violations of these assumptions. The reportedWilcoxon rank-sum test p-values were based on the exact distribution ofthe test statistic.

For completeness, pairwise comparisons were also performed using theunequal variances t test. We used the unequal variances t procedurerather than the equal variances t procedure since ratios of the standarddeviations of the samples varied by as much as 3 to 4 in some cases.

The primary efficacy analysis was comparisons between doxycycline aloneand doxycycline+misoprostol in mean changes in pocket depths at 52 weekspost-treatment. No multiple-comparison correction was used, since thesewere considered to be a priori hypotheses and there were only 7 a priorihypotheses.

All other pairwise comparisons were considered secondary analyses. Thesecondary analyses were considered to be exploratory in nature, andmultiple-comparison correction procedures were not used in order todetect all clinically meaningful differences. However, we recognize thatconclusions drawn from these analyses must be verified by futureresearch.

1.7.1.2 Safety Analyses No formal analyses were performed on any of thesafety parameters.

1.7.2 Determination of Sample Size In a previous study (Hill, R W,Ramfjord, S P, Morrison, E C, Appleberry, E A, Caffesse, R G, Kerry, GJ, and Nissle, R R., Four types of periodontal treatment compared overtwo years, J. Periodontol 51981, 2: 655 (1981)), patients receivingscaling and root planing only and having pockets greater than or equalto 7 mm had a mean change in pocket reduction from baseline of 2.85 mmwith a standard deviation of 1.91 mm after one year. Since similarpatients treated herein with Cipro® (ciprofloxacin) & Flagyl®(metronidazole) plus misoprostol with scaling and root planing treatmentshowed an average exceeding 5.85 mm, we used 3 mm as our effect size, wewould need 10 patients in each group in order to achieve a power of 0.9to detect a 3 mm difference between treatments; this computation isbased on a two-sample t test with a 0.05 two-sided significance leveland assumes a common standard deviation of 1.91. This computation wasperformed using the nQuery Advisor 4.0 software. In order to allow fordropouts and for variability in the mean reduction and standarddeviation estimates, the sample size was increased to 13.

2.0 Study Patients

2.1 Disposition of Patients In an initial study, a total of 58 patientswere enrolled; 58 patients were remaining in the study at 6 weekspost-treatment and 40 patients were remaining in the study at 52 weekspost-treatment.

2.2 Protocol Deviations In the initial study, Patient 51, who wasrandomized to receive placebo+misoprostol, mistakenly receivedplacebo+placebo. The error occurred at the University of Iowa DentalSchool Pharmacy. The pocket depth data for this patient showed much lessimprovement than other patients in the placebo+misoprostol group.However, the dosing error was not discovered until after the statisticalanalysis had been performed and, therefore, all data for this patientwere included in the statistical analysis for the placebo+misoprostolgroup.

3.0 Efficacy Evaluation

3.1 Data Sets Analyzed All available data were used in the efficacysummaries (N=58 at six weeks and N=40 at 52 weeks).

TABLE 2 Patients Excluded from the Efficacy Analyses Doxycycline + Miso-Assessment Placebo Doxycycline Misoprostol prostol 52 Weeks Post- 1, 32,10, 21, 54, 14, 22, 40, 28, 35, treatment 33, 64 60, 70 50, 58 59, 71

3.2 Demographics and Other Baseline Characteristics Enrolled patientsranged in age from 34 to 77 years, and mean ages ranged from 50.1 to53.4 years across the treatment groups (Table 3). Patients wereprimarily Caucasian (≧85.7% in each treatment group), and except for thedoxycycline+misoprostol group, gender was fairly evenly distributed. Allof the patients had a Class III AAP diagnosis.

TABLE 3 Demographics and Baseline Characteristics Doxycycline + PlaceboDoxycycline Misoprostol Misoprostol P-Value N = 14 N = 15 N = 15 N = 14(a) Age (yr) Mean ± SD 51.9 ± 10.58 50.1 ± 7.07 50.2 ± 8.95 53.4 ± 7.100.12 Range 37-77 34-66 37-67 40-65 Gender (N, %) Males 8 (57.1%) 6(40.0%) 12 (80.0%) 6 (42.9%) 0.69 Females 6 (42.9%) 9 (60.0%) 3 (20.0%)8 (57.1%) Race (N, %) White 12 (85.7%) 15 (100.0%) 13 (86.7%) 14(100.0%) Black 2 (14.3%) 0 (0.0%) 1 (6.7%) 0 (0.0%) Unknown 0 (0.0%) 0(0.0%) 1 (6.7%) 0 (0.0%) AAP Diagnosis (N, %) Class III 14 (100.0%) 15(100.0%) 15 (100.0%) 14 (100.0%) Class IV 0 (100.0%) 0 (100.0%) 0(100.0%) 0 (100.0%) (a) ANOVA for comparison of age and Pearsonchi-square test for comparison of proportion of males.

3.3 Measurements of Treatment Compliance No quantitative measurements oftreatment compliance were performed. All patients were provided four10.0 ml syringes of study medication containing sufficient quantities ofstudy medication for the six-week post-treatment period. Patients werequeried at one, two and six weeks post-treatment concerningadministration of the study medication. However, 10 patients reportedthat they did not have sufficient medication for the entire six-weekpostdosing interval and were provided additional syringes of misoprostolpaste (Table 4).

TABLE 4 Additional Study Medication Treatment (a) Patient AdditionalSyringes A 33 2 A 63 1 A 68 2 B 49 2 B 65 2 C 6 2 C 40 2 D 35 2 D 46 2 D71 2

3.4 Efficacy Results and Tabulations of Individual Patient Data

3.4.1 Efficacy Results Descriptive statistics for the change in pocketdepth after 6 and 52 weeks for each group and baseline depth aredisplayed in the end-of-text Tables 1 and 2 and Table 5. Plots of themean changes per observation and per patient are displayed respectivelyin FIGS. 1A and 1B (6 weeks) and in FIGS. 1C and 1D (52 weeks).

3.4.1.1 Pocket Depth Changes Mean changes in pocket depth weredetermined for each observation for each patient. Similar results wereobtained using the two methods (FIGS. 1A-1D). At six weekspost-treatment, all three active treatments had a greater effect onpocket depth than placebo; however, at 52 weeks post-treatment, themisoprostol groups (placebo+misoprostol and doxycycline+misoprostol) hadgreater effects than doxycycline+placebo or placebo.

Due to a dosing error, one patient (51) in the placebo+misoprostol groupreceived placebo+placebo. Since the statistical analysis was performedusing an intent-to-treat cohort, all data for this patient were includedin the summary statistics and statistical analyses for theplacebo+misoprostol group.

For the primary comparison between doxycycline+placebo anddoxycycline+misoprostol, administration of doxycycline+misoprostolresulted in statistically significantly greater mean changes in pocketdepths compared to doxycycline+placebo at 52 weeks post-treatment forpockets having baseline depths between ≦4 and 9 mm (Table 5).

TABLE 5 Summary of Mean Changes in Pocket Depths Using Patient asInference ≦4 mm 5 mm 6 mm 7 mm 8 mm 9 mm 10 mm 6 Weeks Placebo 0.56 1.341.72 1.99 2.39 2.79 3.29 N = 1433/14 N = 386/14 N = 165/14 N = 127/14 N= 133/13 N = 38/11 N = 31/8 Doxycycline 0.84  1.96*  2.63*  3.04*  3.57* 4.23* 4.13 N = 1292/15 N = 393/15 N = 244/15 N = 237/15 N = 149/15 N =52/12 N = 32/9 Doxycycline + 0.85  1.85*  2.49*  3.30*  3.81* 4.04 4.51Misoprostol N = 1533/15 N = 385/15 N = 176/15 N = 161/15 N = 142/14 N =45/13  N = 24/12 Misoprostol 0.58 1.57 2.13 2.68 2.84  4.46* 4.70 N =1573/14 N = 242/14 N = 130/14 N = 97/13  N = 86/14  N = 29/11  N = 19/1052 Weeks Placebo 0.10 0.86 1.43 1.77 1.90 2.22 3.18 N = 993/10  N =286/10 N = 131/10 N = 105/10 N = 95/9  N = 29/7  N = 28/6 Doxycycline−0.22* 0.42 0.79 1.42 1.71 2.86 3.28 N = 819/10  N = 275/10 N = 150/10 N= 154/10 N = 103/10 N = 46/10 N = 25/5 Doxycycline +    1.13*&†  2.31*† 2.98*†  4.04*†  4.89*†  5.07*† 5.52 Misoprostol N = 970/10  N = 270/10N = 130/10 N = 123/10 N = 117/10 N = 35/10 N = 13/8 Misoprostol  0.75*† 1.76*†  2.49†  3.28*†  3.90*†  5.06*† 4.75 N = 1231/10 N = 145/10 N =73/10  N = 58/9  N = 62/10  N = 23/9  N = 11/6 *p < 0.05 compared toplacebo using Wilcoxon Rank-Sum test. &p < 0.05 compared to misoprostolusing Wilcoxon Rank-Sum test. †p < 0.05 compared to doxycycline usingWilcoxon Rank-Sum test. N given as Number of observations/Number ofpatients

At six weeks post-treatment, there were no statistically significantdifferences between the active treatment groups for the mean changes inpocket depths. Both doxycycline+placebo and doxycycline+misoprostol werestatistically superior to placebo in the majority of the assessments(for baseline depths between ≦4 mm through 9 mm for doxycycline+placeboand between ≦4 mm through 8 mm for doxycycline+misoprostol).

At 52 weeks post-treatment, the only assessment showing statisticalsignificance between doxycycline and placebo was for pocket depths ≦4mm. As stated above, doxycycline+misoprostol showed statisticallysignificantly greater changes in mean pocket depths compared todoxycycline+placebo in all assessments except for those having abaseline depth of 10 mm. Similar results were obtained whendoxycycline+misoprostol was compared to placebo. Whenplacebo+misoprostol was administered, mean changes in pocket depths werestatistically superior to placebo and doxycycline treatments in themajority of the assessments. Although mean changes in pocket depths withdoxycycline+misoprostol were numerically superior to those forplacebo+misoprostol, only one (≦4 mm) of the assessments wasstatistically significantly different between treatments.

For baseline pocket depths ≦10 mm, 11 of 28 samples had a skewnessexceeding 1 in magnitude and 10 samples have a kurtosis exceeding 2,suggesting that the normality assumption was not tenable (end-of-textTable 2). End-of-text FIGS. 3 and 4 did not reveal any seriousviolations of the Wilcoxon assumptions.

3.4.1.1.1 Effect of Doxycycline The graphs in FIG. 1 show that 6 weeksand 52 weeks after the scaling, root planning and curettage procedure,the patients showed a reduction in pocket depth, regardless of how deepthe pocket was originally. Even in the placebo treatment group, pocketreductions were in the order of 25-30%, indicating that this basicapproach to treatment is beneficial, even though not as optimal asdesired.

The graphs in FIGS. 1A-1B show that 6 weeks after the scaling, rootplaning and curettage treatment session, the patients that werepretreated with doxycycline (with or without misoprostol treatment) havea greater decrease in their pocket depths than did patients receivingplacebo. They also appeared to have a greater decrease in the pocketdepths than did the patients receiving only misoprostol, except possiblywhen the original pockets depths were 10 mm and greater. (Seeend-of-text Table 1 for results on all pocket depths, including 12 mmand 15 mm.) The importance of the doxycycline pre-treatment on the6-week status of the patients is consistent whether one looks at thedata by observation site, or by the patient (which effectively combinesthe results of several observation sites).

As shown in FIGS. 1C and 1D, the added benefit of the doxycyclinepre-treatment, while very apparent 6 weeks after stopping the treatment(FIGS. 1A and 1B), was not apparent 52 weeks after the scaling, rootplaning and curettage. Without an accompanying misoprostol treatmentregimen, doxycycline pre-treatment did not have any additional long-termbenefit in the control of the patients' periodontal disease beyond thatof the standard scaling, root planing and curettage alone (placebo). Acomparison between the Figures at 6 weeks (FIGS. 1A and 1B) and at 52weeks (FIGS. 1C and 1D) show that part of the early benefit obtainedwith doxycycline was actually lost during the intervening 46 weeks. Forinstance, places were the pocket depth was originally 10 mm, showed areduction in depth of nearly 5 mm at 6 weeks, but that improvement wasnot permanent, reverting to only a 3 mm improvement by Week 52.

3.4.1.1.2 Effect of Topical Misoprostol The two treatment groups thatreceived misoprostol showed additional improvement beyond that provideby scaling, root planing and curettage alone. When examined 6 weeksafter the scaling, root planing and curettage and after 6 weeks of localmisoprostol administration, the misoprostol alone treatment appeared tobe more beneficial as the pocket depth increased. In cases where theoriginal pocket depth was 10 mm or greater, the reduction in pocketdepth after 6 weeks was by 40-50%, and remained there even through tothe one year observation point. The misoprostol applications werediscontinued at the 6- or 7-week time point by the patients.

In patients that had the doxycycline pre-treatment before the scaling,root planing and curettage, and used the misoprostol applications for6-7 weeks afterwards, the benefits were maximal among the treatmentsinvestigated. At the 6-week observation, the patients receiving the dualregimen performed as well as the doxycycline only patients. At the52-week observation the patients receiving the dual regimen showed thegreatest reduction in pocket depth of all groups. The dual regimen wasthe only treatment that resulted in a trend towards apparent continuedimprovement in pocket depth between the Week 6 and Week 52 observations.These patients had approximately 40-50% mean reductions in pocket depthat Week 6, and approximately 50-60% percent mean reduction at Week 52.

3.4.1.1.3 Effect on Pocket Depth of Topical Misoprostol and Doxycyclineat 3 Weeks Post Treatment Versus Topical Misoprostol alone after 6 WeeksPost Treatment In a larger study, the change in pocket depth wasdetermined for those patients presenting the inclusion criteria ofExample 1.3.1 and none of the exclusion criteria of Example 1.3.2 afterthree (3) weeks of treatment with misoprostol and doxycycline (newdata), as described herein, and was compared to the change in pocketdepth at 6 weeks for those patients also treated with misoprostol anddoxycycline (Group C patients of Example 1). The comparative data inTable 6 is reported as a function of the patient's baseline pocket depth(2 mm to 15 mm) prior to treatment. Table 6 reports each of thefollowing: the number of observations (N_Obs), the mean change in pocketdepth for all surfaces for all patients (i.e., all observations) havingthe specified baseline pocket depth (“mean of observation”), the numberof subjects (N_subj), and the mean of the individual means (“mean ofsubject means”) wherein an individual mean is the mean change for allsurfaces for that individual having the specified baseline depth.

Based upon the Wilcoxon p-value, Table 6 reflects that the combinationof misoprostol and doxycycline produced a statistically significantchange (improvement) in pocket depth at three weeks versus 6 weeks forthe subject patients.

TABLE 6 Change in pocket depth (mm) after 3 weeks topical cotreatmentwith misoprostol/antibiotic (“new data”) compared with change after 6weeks topical treatment with misoprostol (Group “C” herein) Mean Meanchange change baseline (mm) of (mm) of depth obser- subject Wilcoxon(mm) group N_Obs vations N_subj means p_value¹ ≦2 C 71 −0.03 6 −0.030.4893 ≦2 New Data 2863 −0.01 34 −0.01 3 C 973 0.67 15 0.66 0.1710 3 NewData 1491 0.75 33 0.75 4 C 509 1.34 15 1.29 0.0876 4 New Data 829 1.5931 1.52 5 C 385 1.89 15 1.85 0.0201 5 New Data 667 2.31 32 2.21 6 C 1762.53 15 2.49 0.0468 6 New Data 315 2.99 33 2.86 7 C 161 3.39 15 3.300.3027 7 New Data 139 3.61 29 3.50 8 C 142 3.70 14 3.81 0.2111 8 NewData 95 4.28 24 4.16 9 C 45 4.20 13 4.04 0.1117 9 New Data 35 4.97 154.96 10 C 24 4.63 12 4.51 0.1529 10 New Data 51 5.84 13 5.69 12 C 196.53 7 5.32 0.0455 12 New Data 37 7.11 12 7.70 15 C 9 8.44 5 7.70 0.722815 New Data 6 8.83 4 9.13 ¹The Wilcoxon p-value is based on the subjectmeans.

4.0 Oral Pastes

4.1 Misoprostol Oral Paste (10 μg/0.15 g paste). In a stock jar, 20.001g of ground CYTOTEC® tablets (containing 20.0 mg of misoprostol) wascombined with 114.999 g of SUPER WERNET'S® Denture Adhesive Powder. Thecombination was mixed well by shaking. To the mixture was added 165 g ofBase A/C solution. The Base AC solution comprises 5% polyethylene glycolMW 1450 (PEG 1450) and 95% polyethylene glycol MW 300 (PEG 300). Thewetted mixture was stirred to break up any lumps and to remove thepowder off the side of the container. The mixture was then allowed toset overnight to fully wet. The following morning, the mixture wasstirred with a metal spatula to produce 300 g of 0.00667% misoprostoloral paste (10 μg misoprostol/150 mg oral paste). The resulting pastewas then dispensed in 10 g aliquots (approximately 8.8 ml) into a 10 mlsyringe suitable for oral application. In this embodiment and those thatfollow, the 150 mg of oral paste corresponds to 0.1275 ml (0.13 ml) ofpaste in the syringe.

4.2 Misoprostol (0.00667%)/Doxycycline (1%) Oral Paste. In a stock jar,20.001 g of ground CYTOTEC® tablets (containing 20.0 mg of misoprostol)was combined with 111.999 g of SUPER WERNET'S® Denture Adhesive Powderand 3.0 g of doxycycline. The combination was mixed well by shaking. Tothe mixture was added 165 g of Base A/C solution (5% PEG 1450:95% PEG300 wt/wt). The wetted mixture was stirred to break up any lumps and toremove the powder off the side of the container. The mixture was thenallowed to set overnight to fully wet. The following morning, themixture was stirred with a metal spatula to produce 300 g of 0.00667%misoprostol/1% doxycycline oral paste (10 μg misoprostol and 1.5 mgdoxycycline/150 mg oral paste). The resulting paste was then dispensedin 10 g aliquots (approximately 8.8 ml) into a 10 ml syringe suitablefor oral application.

4.3 Misoprostol (0.00667%)/Gentamicin (1%) Oral Paste. In a stock jar,20.001 g of ground CYTOTEC® tablets (containing 20.0 mg of misoprostol)was combined with 111.999 g of SUPER WERNET'S® Denture Adhesive Powderand 3.0 g of gentamicin. The combination was mixed well by shaking. Tothe mixture was added 165 g of Base A/C solution (5% PEG 1450:95% PEG300 wt/wt). The wetted mixture was stirred to break up any lumps and toremove the powder off the side of the container. The mixture was thenallowed to set overnight to fully wet. The following morning, themixture was stirred with a metal spatula to produce 300 g of 0.00667%misoprostol/1% gentamicin oral paste (10 μg misoprostol and 1.5 mggentamicin/150 mg oral paste). The resulting paste was then dispensed in10 g aliquots (approximately 8.8 ml) into a 10 ml syringe suitable fororal application.

4.4 Misoprostol (0.00667%)/Clarithromycin (1%) Oral Paste. In a stockjar, 20.001 g of ground CYTOTEC® tablets (containing 20.0 mg ofmisoprostol) was combined with 111.999 g of SUPER WERNET'S® DentureAdhesive Powder and 3.0 g of clarithromycin. The combination was mixedwell by shaking. To the mixture was added 165 g of Base A/C solution (5%PEG 1450:95% PEG 300 wt/wt). The wetted mixture was stirred to break upany lumps and to remove the powder off the side of the container. Themixture was then allowed to set overnight to fully wet. The followingmorning, the mixture was stirred with a metal spatula to produce 300 gof 0.00667% misoprostol/1% clarithromycin oral paste (10 μg misoprostoland 1.5 mg clarithromycin/150 mg oral paste). The resulting paste wasthen dispensed in 10 g aliquots (approximately 8.8 ml) into a 10 mlsyringe suitable for oral application.

4.5 Misoprostol (0.00667%)/Azithromycin (1%) Oral Paste. In a stock jar,20.001 g of ground CYTOTEC® tablets (containing 20.0 mg of misoprostol)was combined with 111.999 g of SUPER WERNET'S® Denture Adhesive Powderand 3.0 g of azithromycin. The combination was mixed well by shaking. Tothe mixture was added 165 g of Base A/C solution (5% PEG 1450:95% PEG300 wt/wt). The wetted mixture was stirred to break up any lumps and toremove the powder off the side of the container. The mixture was thenallowed to set overnight to fully wet. The following morning, themixture was stirred with a metal spatula to produce 300 g of 0.00667%misoprostol/1% azithromycin oral paste (10 μg misoprostol and 1.5 mgazithromycin/150 mg oral paste). The resulting paste was then dispensedin 10 g aliquots (approximately 8.8 ml) into a 10 ml syringe suitablefor oral application.

4.6 Misoprostol (0.00667%)/Clindamycin (1%) Oral Paste. In a stock jar,20.001 g of ground CYTOTEC® tablets (containing 20.0 mg of misoprostol)was combined with 111.999 g of SUPER WERNET'S® Denture Adhesive Powderand 3.0 g of clindamycin. The combination was mixed well by shaking. Tothe mixture was added 165 g of Base A/C solution (5% PEG 1450:95% PEG300 wt/wt). The wetted mixture was stirred to break up any lumps and toremove the powder off the side of the container. The mixture was thenallowed to set overnight to fully wet. The following morning, themixture was stirred with a metal spatula to produce 300 g of 0.00667%misoprostol/1% clindamycin oral paste (10 μg misoprostol and 1.5 mgclindamycin/150 mg oral paste). The resulting paste was then dispensedin 10 g aliquots (approximately 8.8 ml) into a 10 ml syringe suitablefor oral application.

4.7 Misoprostol (0.00667%)/Metronidazole (1%) Oral Paste. In a stockjar, 20.001 g of ground CYTOTEC® tablets (containing 20.0 mg ofmisoprostol) was combined with 111.999 g of SUPER WERNET'S® DentureAdhesive Powder and 3.0 g of triturated metronidazole. The combinationwas mixed well by shaking. To the mixture was added 165 g of Base A/Csolution (5% PEG 1450:95% PEG 300 wt/wt). The wetted mixture was stirredto break up any lumps and to remove the powder off the side of thecontainer. The mixture was then allowed to set overnight to fully wet.The following morning, the mixture was stirred with a metal spatula toproduce 300 g of 0.00667% misoprostol/1% metronidazole oral paste (10 μgmisoprostol and 1.5 mg metronidazole/150 mg oral paste). The resultingpaste was then dispensed in 10 g aliquots (approximately 8.8 ml) into a10 ml syringe suitable for oral application.

4.8 Misoprostol (00667%)/Ciprofloxacin (1%) Oral Paste. In a stock jar,20.001 g of ground CYTOTEC® tablets (containing 20.0 mg of misoprostol)was combined with 111.999 g of SUPER WERNET'S® Denture Adhesive Powderand 3.0 g of ciprofloxacin hydrochloride (ciprofloxacin). Thecombination was mixed well by shaking. To the mixture was added 165 g ofBase A/C solution (5% PEG 1450:95% PEG 300 wt/wt). The wetted mixturewas stirred to break up any lumps and to remove the powder off the sideof the container. The mixture was then allowed to set overnight to fullywet. The following morning, the mixture was stirred with a metal spatulato produce 300 g of 0.00667% misoprostol/1% ciprofloxacin oral paste (10μg misoprostol and 1.5 mg ciprofloxacin/150 mg oral paste). Theresulting paste was then dispensed in 10 g aliquots (approximately 8.8ml) into a 10 ml syringe suitable for oral application.

1. A pharmaceutical composition comprising in combination: (i) atherapeutically effective amount of misoprostol in the amount of0.00667%, or a pharmaceutically acceptable acid or salt thereof; (ii) aneffective amount of ciprofloxacin in the amount of 1%; and (iii) apharmaceutically acceptable carrier suitable for topical application. 2.The pharmaceutical composition of claim 1, wherein said therapeuticallyeffective misoprostol, or pharmaceutically acceptable acid or saltthereof is in the form of a racemate, enantiomer, or diastereomer. 3.The pharmaceutical composition of claim 1, wherein said misoprostol, ora pharmaceutically acceptable acid or salt thereof is misoprostolicacid.
 4. The pharmaceutical composition of claim 1, further comprisingat least one colloidal dispersion system.
 5. The pharmaceuticalcomposition of claim 4, wherein said colloidal dispersion systemcomprises carbowax.
 6. The pharmaceutical composition of claim 1,further comprising at least one additive or preservative.
 7. Thepharmaceutical composition of claim 6, wherein said additive orpreservative comprises polyethylene glycol having an average molecularweight ranging from about 300 MW to about 1450 MW.
 8. The pharmaceuticalcomposition of claim 1, further comprising at least one dentureadhesive.
 9. The pharmaceutical composition of claim 8, wherein saiddenture adhesive is in powdered form.
 10. The pharmaceutical compositionof claim 1, wherein said pharmaceutically acceptable carrier is a slowrelease agent.
 11. The pharmaceutical composition of claim 1, whereinsaid pharmaceutical composition is in the form of a topical or oralformulation.
 12. The pharmaceutical composition of claim 11, whereinsaid topical formulation is an oral paste.
 13. A method for treatingperiodontal disease in a mammalian patient comprising: (a) administeringto, into, on at or in a periodontal pocket of said patient in need oftreatment for periodontal disease a therapeutically effective amount ofa pharmaceutical composition comprising 0.00667% misoprostol, or apharmaceutically acceptable acid or salt thereof in a pharmaceuticallyacceptable carrier; (b) administering to, into, on at or in aperiodontal pocket of said patient an effective amount of apharmaceutical composition comprising 1% ciprofloxacin in apharmaceutically acceptable carrier; (c) optionally, administering tosaid patient prior to step (a) or step (b) or both, at least one step ofsub-gingival scaling, root planning, or curettage; whereby saidperiodontal disease in said mammalian patient is ameliorated.
 14. Themethod of claim 13, wherein said mammalian patient is human.
 15. Themethod of claim 13, wherein said mammalian patient is a domesticatedanimal.